Increased understanding of surface-enhanced Raman scattering (SERS) has expanded the utility of Raman spectroscopy for a variety of applications requiring a high degree of chemical specificity. In recent years, SERS has shown tremendous potential as a powerful and ultrasensitive detection technique at the trace and even the single molecule level. One of the benefits of SERS detection is the ability to probe the structural properties of compounds in various physical environments. More particularly, the chemical specificity and insensitivity to water render SERS an ideal candidate for highly sensitive detection of analytes in aqueous environment.
High sensitivity SERS detection in flow has remained challenging. SERS originates from molecules located in close proximity to metallic nanostructures that are capable of generating a localized surface plasmon resonance (LSPR). As a result, one of the inherent requirements for SERS signal generation is that molecules must be located near the enhancing surface. This distance dependence intrinsic to SERS varies based on the type of nanostructures used for the SERS substrate. For individual nanoparticles the enhancement extends a few nanometers whereas an exponential decay of the evanescent field with a length scale of ˜10 nm is observed on extended surfaces. Traditionally, depositing a solution onto a metallic nanostructure and allowing it to evaporate adsorbs molecules to the surface. In solution, however, the ability of molecules to diffuse away from the nanostructures results in limited sensitivity. It follows that the number of molecules present in the enhanced region in dilute solution is often below the limit of detection. These effects typically require micromolar or greater solution concentrations.
Nanostructure-analyte interactions in the SERS detection volume are key to improving signal sensitivity. A common approach used to promote this interaction involves mixing of the sample analyte and the colloids, either directly in a microfluidic channel or off-line prior to being introduced in the fluidic system. These techniques can achieve high sensitivity and are known to reduce problems associated with variations in sample mixing, localized heating, and photodissociation. However, the major drawbacks of using metal colloids for SERS-based assays are their lack of chemical affinity for the target analyte in solution and problems associated with non-specific adsorption that complicate detection. The random aggregation of nanoparticles is also known to affect the reproducibility of the acquired SERS spectrum. Under these conditions, SERS measurements are recorded using extended acquisition times greater or equal to one second to improve limits of detection, but limiting throughput.
Two dimensional planar substrates avoid many complications associated with nanoparticles. However, the limit of detection of 2-D substrates in solution is still controlled by transport, which can hinder analyte interaction with the SERS-active surface. Over the years, methods have been developed to increase substrate-analyte interactions. Chemical modifications have been shown to increase affinity of the analyte molecules for the SERS substrate. Such techniques concentrate nanoparticle-analyte conjugates in the detection volume to improve detection, but are limited to analytes with high affinity for the functionalized surface. Other techniques actively concentrate nanoparticle-analyte conjugates into the detection volume using electrokinetic or magnetic forces. Although these techniques improve sensitivity and the functionality of the SERS assays, the incorporation of active elements or additional fabrication steps add cost and complexity to the final device. Despite the recent advances in performance and sensitivity, these inherent drawbacks limit the successful translation of SERS from the research lab to practical applications.
Accordingly, devices and methods are needed that enable the confinement of a sample fluid near a detection surface to promote interaction between sample molecules and a SERS substrate, thereby increasing the performance and sensitivity of these analytical techniques.